Human hepatoma cell (HepG2) monolayer cultures has previously been shown to synthesize and secrete thyroxine-binding globulin (TBG). The present studies employed these cells to demonstrate that 27 K protein, a novel thyroxine-binding protein described in Project Z01 AM 45000-18, is also produced by the liver. Convincing evidence that TBG and 27 K protein are distinct proteins was obtained by showing that TBG mRNA and 27 K mRNA can be separated by sucrose gradient centrifugation. In vitro translation of 27 K mRNA and whole cell studies with (H3)mannose as precursor, or (S35)methionine in the presence of the glycosylation inhibitor, tunicamycin, proved that 27 K protein is nonglycosylated and does not appear to contain a pro-peptide. The kinetics of 27 K protein secretion was similar to that of albumin and faster than that of TBG, reflecting its non-glycoprotein nature. Earlier studies with monkey hepatoma cell cultures showed that 27 K protein was a much more prominent biosynthetic product than TBG in these cells.